Arrangements and methods for providing multimodality microscopic imaging of one or more biological structures

ABSTRACT

Method and apparatus according to an exemplary embodiment of the present invention can be provided. For example, first data associated with a first signal received from at least one region of at least one sample can be provided based on a first modality, and second data associated with a second signal received from the at least one sample can be provided based on a second modality which is different from the first modality. Third data associated with a reference can be received. Further data can be generated based on the first, second and third data. In addition, third data associated with a second signal received from the at least one sample can be obtained. Each of the third data can be based on a further modality which is different from the first modality and the second modality, and the further data can be further determined based on the third data. Further, the first modality can be a spectral-encoded modality, and the second modality can be a non-spectral-encoding modality.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This application is based upon and claims the benefit of priority fromU.S. patent application Ser. No. 60/721,802, filed Sep. 29, 2005, theentire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention generally relates to arrangements and methods forproviding multimodality microscopic imaging of one or more biologicalstructures, and particularly to, e.g., conducting reflectance and/orfluorescence microscopy of biological specimens using spectrally encodedconfocal microscopy (“SECM”), fluorescence SECM, optical coherencetomography (“OCT”), spectral domain (“SD”)-OCT, optical frequency domaininterferometry (“OFDI”), and optical coherence microscopy (“OCM”)procedures.

BACKGROUND OF THE INVENTION

A determination of the relationship between the molecular basis ofgenetic alterations and phenotype generally utilizes accurate two- andthree-dimensional characterization of microstructure of biologicalspecimens. However, motion and small dimensions make many livingbiological specimens can be more difficult to evaluate.

Optical techniques offer the potential to image the biological specimensat a high resolution. For certain applications, optical imaging based onendogenous contrast can be advantageous over techniques that requireexogenous agents, since such beneficial procedures can allow theanalysis of the specimen in its native state and at multiple timepoints, with a small amount of preparation. As an example, severalendogenous-contrast imaging modalities are described herein forvisualizing embryonic heart microstructure: two exemplary forms ofoptical coherence tomography (“OCT”) as described in D. Huang et al.,“Optical coherence tomography,” Science 254, pp. 1178-1181 (1991),time-domain optical coherence tomography (“TD-OCT”) as described in S.A. Boppart et al., “Investigation of developing embryonic morphologyusing optical coherence tomography,” Dev Biol 177, pp. 54-63 (1996), andoptical frequency domain imaging (“OFDI”) as described in M. A. Choma etal., “Sensitivity advantage of swept source and Fourier domain opticalcoherence tomography,” Optics Express 11, pp. 2183-2189 (2003); and S.H. Yun et al., “High-speed optical frequency-domain imaging,” OpticsExpress 11, pp2953-2963 (2003).

Additional examples can be provided and utilized including tworeflectance microscopy techniques, e.g., full-field optical coherencemicroscopy (“FFOCM”) as described in E. Beaurepaire et al., “Full-fieldoptical coherence microscopy,” Optics Letters 23, pp. 244-246 (1998); A.Dubois et al., “Ultrahigh-resolution full-field optical coherencetomography,” Appl Opt 43, pp. 2874-2883 (2004); and G. Moneron et al.,“Stroboscopic ultrahigh-resolution full-field optical coherencetomography,” Opt Lett 30, pp. 1351-1353 (2005), and spectrally encodedconfocal microscopy (“SECM”) as described in G. J. Tearney et al.,“Spectrally encoded confocal microscopy,” Optics Letters 23, pp.1152-1154 (1998); and C. Boudoux et al., “Rapid wavelength-sweptspectrally encoded confocal microscopy,” Optics Express 13, pp.8214-8221 (2005).

For example, the TDOCT techniques can use low-coherence interferometryto obtain cross-sectional images with ˜10 μm resolution and at depths ofup to 2 mm. (See S. A. Boppart et al., “Noninvasive assessment of thedeveloping Xenopus cardiovascular system using optical coherencetomography,” Proc Natl Acad Sci U S A 94, pp. 4256-4261 (1997); S.Yazdanfar et al., “High resolution imaging of in vivo cardiac dynamicsusing color Doppler optical coherence tomography,” Optics Express 1, pp.424-431 (1997); T. M. Yelbuz et al., “Optical coherence tomography: anew high-resolution imaging technology to study cardiac development inchick embryos,” Circulation 106, pp. 2771-2774 (2002); V. X. D. Yang etal., “High speed, wide velocity dynamic range Doppler optical coherencetomography (Part II): Imaging in vivo cardiac dynamics of Xenopuslaevis,” Optics Express 11, pp. 1650-1658 (2003); and W. Luo et al.,“Three-dimensional optical coherence tomography of the embryonic murinecardiovascular system ” Journal of biomedical optics 11, 021014 (2006).

The exemplary OFDI technique can be considered as a derivative of theTDOCT techniques that may enable an acquisition of images atsignificantly higher frame rates as described in R. Huber et al.,“Three-dimensional and C-mode OCT imaging with a compact, frequencyswept laser source at 1300 nm,” Optics Express 13, pp. 10523-10538(2005). The high speed of the OFDI techniques can enable animplementation of a true four-dimensional (4D) microscopy (e.g.,three-dimensional microscopy as a function of time). Full-field opticalcoherence microscopy (“FFOCM”) techniques can utilize low-coherenceinterferometry and higher numerical aperture objective lenses to attainresolution at the subcellular level in all three dimensions. Such FFOCMtechniques are likely considerably slower than the OFDI techniques. Theexemplary SECM techniques can have a form of the reflectance confocalmicroscopy using which it may be possible to obtain two-dimensionalimages with micron-level resolution, at significantly higher speeds thanpossibly obtained using the FFOCM techniques.

While each of these natural-contrast procedures can individually be usedfor imaging a microstructure of the embryonic heart, when combined,these procedures can provide a powerful set of tools for two-, three-,and four-dimensional characterization of early myocardial morphology anddynamics. A combination of these different modalities into one singlemicroscopy device may have additional advantages such as, e.g., (a) acomparison of images in different formats, different resolutions, andfields of view, (b) a simultaneous acquisition of both structural andfunction information, and/or (c) these tasks can be accomplished usingone instrument without requiring moving or altering the specimen.

OBJECTS AND SUMMARY OF THE INVENTION

One of the objects of the present invention is to overcome certaindeficiencies and shortcomings of the prior art systems (including thosedescribed herein above), and provide exemplary embodiments of providingmultimodality microscopic imaging of one or more biological structures.Such exemplary embodiments can conduct reflectance and/or fluorescencemicroscopy of biological specimens using spectrally encoded confocalmicroscopy (“SECM”), fluorescence SECM, optical coherence tomography(“OCT”), spectral domain (“SD”)-OCT, optical frequency domaininterferometry (“OFDI”), and optical coherence microscopy (“OCM”)procedures.

For example, an analysis of biological specimens generally employs avisualization of its microstructure and functions, preferably with smallalterations to the specimen. According to one exemplary embodiment ofthe present invention, a combination of multiple different imagingmodalities can be provided in a single microscope device. Each exemplarytechnique according to certain exemplary embodiments of the presentinvention can provide distinct and complementary imaging capabilities,including high-speed (e.g., 1000 frames per second) and high axialresolution (4-16 μm) cross-sectional imaging in vivo, truefour-dimensional imaging in vivo, three-dimensional microscopy withisotropic cellular (e.g., 1-2 μm) resolution in vitro, andtwo-dimensional subcellular imaging in vivo. When combined, theseexemplary imaging modalities can effectuate a more complete picture ofthe morphologic and dynamics of biological specimens.

Thus, the exemplary embodiments of the present invention includearrangements and methods for acquiring multimodality microscopic data.For example, according to one exemplary embodiment, it is possible touse a combination of unique broad bandwidth or rapid wavelength sweptsources and optics interposed between a scanning mechanism and animaging lens. Data can be acquired simultaneously and/or serially, e.g.,without moving the specimen. For example, data obtained from differentmodalities can be co-registered so that it can be displayed side-by-sideand/or overlaid on top of each other. Quantitative information can beobtained from all of the datasets in a complementary manner.

Thus, in accordance with the exemplary embodiments of the presentinvention, method and apparatus can be provided. For example, first dataassociated with a first signal received from at least one region of atleast one sample can be provided based on a first modality, and seconddata associated with a second signal received from the at least onesample can be provided based on a second modality which is differentfrom the first modality. Third data associated with a reference can bereceived. Further data can be generated based on the first, second andthird data. In addition, third data associated with a second signalreceived from the at least one sample can be obtained. Each of the thirddata can be based on a further modality which is different from thefirst modality and the second modality, and the further data can befurther determined based on the third data. Further, the first modalitycan be a spectral-encoded modality, and the second modality can be anon-spectral-encoding modality.

In another exemplary embodiment of the present invention, the firstmodality can be florescence imaging. A microscope arrangement and/or abeam-scanning arrangement can be provided. The beam-scanning arrangementmay be configured to forward electro-magnetic radiation to the at leastregion. Further, a two-dimensional image and/or a three-dimensionalimage can be produced as a function of the further data. The first andsecond data may be obtained substantially simultaneously. In addition,the first and second data may be associated with approximately the samelocation on the sample, and/or can be obtained using another one of thefirst and second data.

According to a further exemplary embodiment of the present invention,the apparatus can be provided in a probe and/or a single enclosure. Itis also possible to obtain spectral encoding microscopy informationusing such exemplary apparatus and method, as well as bright field, darkfield, phase contrast, polarization, epireflectance and/or reflectancemicroscopy information. It is further possible to use such exemplaryapparatus and method change from the first modality to the secondmodality. Optical coherence tomography information associated with asignal provided by a source arrangement having a plurality ofwavelengths can be obtained. A plurality of detectors can be provided todetect a spectral interference between the second and third signals as afunction of the wavelengths.

Optical coherence tomography information associated with a signalprovided by a source arrangement can be obtained whose wavelength variesover time. At least one image can be generated based on the first andsecond data. In addition, a first image can be generated based on thefirst data and a second image can be generated based on the second data.The first and second images may be associated with one another as afunction of the first and second data. It is possible to obtain opticalcoherence tomography information and/or optical frequency domaininterferometry information.

Other features and advantages of the present invention will becomeapparent upon reading the following detailed description of embodimentsof the invention, when taken in conjunction with the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further objects, features and advantages of the present invention willbecome apparent from the following detailed description taken inconjunction with the accompanying figures showing illustrativeembodiments of the present invention, in which:

FIG. 1 is a schematic diagram of an exemplary SECM system that utilizesa broad bandwidth source;

FIG. 2 is a schematic diagram of an exemplary SD-OCT system;

FIG. 3 is a schematic diagram of an exemplary OCM system that utilizes abroad bandwidth source;

FIG. 4 is a schematic diagram of an exemplary FFOCM system that utilizesa broad bandwidth source;

FIG. 5 is a schematic diagram of an exemplary fluorescence SECM systemthat utilizes a broad bandwidth source;

FIG. 6 is a schematic diagram of an exemplary SECM system that utilizesa wavelength tuning source;

FIG. 7 is a schematic diagram of an exemplary OFDI system that utilizesa wavelength tuning/modulated source;

FIG. 8 is a schematic diagram of an exemplary OCM system that utilizes awavelength modulated/tuning source;

FIG. 9 is a schematic diagram of an exemplary FFOCM system that utilizesa wavelength modulated/tuning source;

FIG. 10 is a schematic diagram of an exemplary combined SECM/SD-OCT/OCMsystem that utilizes a broad bandwidth source according to a firstexemplary embodiment of the present invention;

FIG. 11 is a schematic diagram of an exemplary combinedSECM/SD-OCT/FFOCM system that utilizes a broad bandwidth sourceaccording to a second exemplary embodiment of the present invention;

FIG. 12 is a schematic diagram of exemplary multimodality microscopesliders according to a particular exemplary embodiment of the presentinvention;

FIG. 13 is a schematic diagram of an exemplary combined SECM/OFDI/OCMsystem that utilizes a wavelength tuning source according to a thirdexemplary embodiment of the present invention;

FIG. 14 is a schematic diagram of an exemplary combined SECM/OFDI/FFOCMsystem that utilizes a wavelength tuning source according to a thirdexemplary embodiment of the present invention;

FIGS. 15 a-15 m are various exemplary images of Xenopus laevis hearts(stage 49) in vivo using exemplary embodiments of the TDOCT and OFDIprocedures.

FIGS. 16 a-16 m are various exemplary three-dimensional images ofXenopus heart in vitro using exemplary embodiments of the FFOCMprocedure.

FIGS. 17 a-17 h are exemplary high-resolution confocal images obtainedin vivo using the exemplary embodiments of the SECM procedure;

FIGS. 18 a-18 e are exemplary images of an aneurismal dilatation in theXenopusheart obtained using the exemplary embodiments of the method andarrangements according to the present invention; and

FIGS. 19 a-19 x are exemplary images of abnormal heart formation due toethanol exposure using the exemplary embodiments of the method andarrangements according to the present invention.

Throughout the figures, the same reference numerals and characters,unless otherwise stated, are used to denote like features, elements,components or portions of the illustrated embodiments. Moreover, whilethe subject invention will now be described in detail with reference tothe figures, it is done so in connection with the illustrativeembodiments. It is intended that changes and modifications can be madeto the described embodiments without departing from the true scope andspirit of the subject invention as defined by the appended claims.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Exemplary SECM techniques are capable of providing subcellular levelresolution images in tissue or biological specimens. SECM images canalternatively represent fluorescence from the sample or reflectance fromthe sample. FIG. 1 depicts a schematic diagram of an exemplary SECMarrangement which utilizes a broadband source. In this exemplaryconfiguration, a quasimonochromatic or broadband light 100 illuminates acirculator 110, which alternatively may be a beam splitter. In oneembodiment this circulator or beam splitter is fiber-optic coupled. Thecore of the optical fiber can serve as the pinhole for the confocalmicroscope system. The fiber may alternatively have multiple claddingsthat transmit light such that for example the light exciting the sampleis single mode and the collected light is multimode. Light from thiselement may be incident on a scanning mechanism 115 that scans the angleof the beam so as to produce one or more transverse scans on the sample.The scanning mechanism may alternatively be one of a resonant scanner,galvanometer scanner, polygon scanning mirror, acousto-optic scanner orthe like. A telescope apparatus may be used to image the scan axis tothe back focal plane of the objective lens 130. Light from the scanningmechanism can then be directed towards a wavelength dispersing element120 such as a transmission diffraction grating, prism, grating prism,dual prism grating prism (DP-GRISM) or the like. This exemplary elementmay disperse the different wavelengths in the broad bandwidth source sothat it is incident on the objective lens 130 with varying angles thatdepend on wavelength.

In one exemplary embodiment, the lens can have a numerical aperture thatmay produce a small focused spot or alternatively the lens has a highNA >0.2. The objective lens 130 focuses each wavelength region onto thesample where each wavelength region on the sample 160 that can belocated at a different spatial location. For a diffraction grating andan objective lens, these exemplary elements may form a wavelengthencoded line 140 on the sample where each position on the line isencoded by a different wavelength region. Light from the sample 160 canbe reflected back through the exemplary system of FIG. 1. Out-of-focuslight may be rejected by the cladding of the optical fiber and in focus(e.g., confocal) light is transmitted back through the circulator/beamsplitter 110 to a spectrometer that measures the spectral content of thereturned light 145. Confocal remittance as a function of spatiallocation is decoded by measuring this spectrum, forming one line on animage. Successive lines are formed for each angular position of thescanning mechanism 115, forming a spectrally-encoded confocal microscopyimage.

FIG. 2 depicts a schematic diagram of an exemplary spectral-domain OCTsystem. Contrary to the exemplary SECM system, the exemplary SD-OCT canprovide cross-sectional images of a biological specimen by usingcoherence gating in the Fourier domain. SD-OCT images can typically havea lower resolution (˜3-10 μm), and may have a larger field of view(several mm's). In this exemplary SD-OCT system, a broad bandwidth orquasimonochromatic source 200 can be input into an interferometer, whichmay be fiber optic-based. The fiber-coupled light can be transmitted toa circulator 210 and a beam splitter 220. When coupled into thecirculator 210, the light can preferably be subsequently split by a beamsplitter 220 so that a portion thereof can be transmitted to a referencearm 225 and a portion is transmitted to a sample arm 235. Light from thereference arm 225 can be reflected off a mirror 230 (e.g., a reference)to the beam splitter 220 or alternatively transmitted back to the beamsplitter 220. In one exemplary embodiment, the splitter 220 can beconfigured so that the majority of light is transmitted to the samplearm 235. Light from the sample arm fiber can then be directed towards alens and a scanning mechanism 240. The scanning mechanism can scan thelight of the sample arm 235 in arbitrary one- or two-dimensionalpatterns. Light can be transmitted from the scanning mechanism to a lens250 which, in one exemplary embodiment, can have a NA so that theconfocal parameter is sufficiently large to allow cross-sectionalimaging in the biological specimen or sample 260.

Light remitted from the sample may be transmitted back through theapparatus to the circulator/beam splitter 210, and directed to aspectrometer 280. The reflectance as a function of depth (A-line) withinthe tissue may be reconstructed by, e.g., a background subtraction,remapping λ-space to k-space, and inverse Fourier transformation of thespectral interference signal in a central processing unit or computer290. Successive A-lines are obtained for each scanning mechanismposition, thereby reconstructing a cross-sectional image of the sample.Alternative exemplary embodiments known in the art, including thecapability to obtain spectral information from the sample byshort-time-Fourier transformation (“STFT”) of the spectral interference,Doppler-sensitive SD-OCT and polarization-sensitive SD-OCT, may be alsoutilized to extract additional information from the biological specimen,such as absorption, flow, and birefringence.

FIG. 3 depicts a schematic diagram of an exemplary optical coherencemicroscopy (“OCM”) system. The exemplary OCM system can utilize acombination of confocal microscopy and OCT techniques that may beadvantageous, as the axial point spread functions of both such exemplarytechniques may be multiplied so as to provide a greater degree ofoptical sectioning. In one exemplary embodiment of the OCM system, lightfrom a broad bandwidth source can be input into a modulating element 310so that the modulation frequency approximates that of the spectralinterference in the interferometer. This exemplary modulation elementmay be one of a Michelson interferometer, pulse shaping apparatus,spectral filter, etc. The modulation may also shift the spectral phaseby some amount over time so that successive spectra may be subtracted toextract only the spectral interference term. Following the modulatingelement, the light can be transmitted to a circulator/beam splitter 320and then, if a circulator is used, to a beam splitter 330. Light canagain be transmitted to a reference arm 335 and a sample arm 345. Lightfrom the reference arm 335 is reflected by a mirror 340. Light from thesample arm 345 can be transmitted to an x-y scanner 350, which canalternatively be one of a or a combination of a resonant scanner,galvanometer scanner, polygon scanning mirror, acousto-optic scanner orthe like.

Light from the scanner 350 can be directed to an objective lens 355 sothat a tightly focused spot can be scanned within the sample. Theobjective lens or sample 360 may be alternatively scanned in any ofthree dimensions to facilitate data collection from different portionswithin the sample. Light is transmitted back from the sample 360 to thecirculator/beam splitter 320 and subsequently to a detection apparatus.In one embodiment, the detector is a spectrometer and OCM data isobtained by obtaining A-lines from the sample in a similar manner asperformed by the exemplary SD-OCT. In the spectral modulation embodimentthe detector can alternatively be a photodiode or other single detectorthat is synchronized to the source modulation element 310. Exemplarylock-in or subtraction techniques can be utilized to extract the OCMsignal.

Full-field optical coherence microscopy is typically a free-spaceinterferometric technique that utilizes a broad bandwidth source toobtain transverse, high-resolution optical sections of biologicalspecimens. FIG. 4A depicts a schematic diagram of an exemplary FFOCMsystem, where broad bandwidth light 400 is transmitted to a beamsplitter 410. Light is split into the sample arm 423 and the referencearm 422. Light in the reference arm 422, according to one exemplaryembodiment, may be directed 415 towards a reference objective lens 420and to a mirror 425, which is capable of an axial motion. Light in thesample arm 423 may be directed towards a sample objective lens 430 andto the sample 440. In one exemplary embodiment, the reference and sampleobjectives 420, 430 have the similar characteristics.

In another exemplary embodiment, the objective lenses 420, 430 may beoptimized for use with immersion fluid that has a refractive index thatis similar to the sample. The sample can be coupled to a stage 443 thatprovides motion in any of three-dimensions. Light from the reference arm422 and the sample arm 423 can be imaged using a lens 445 onto a CCDcamera 450. Fringes are detected by the CCD camera 445 resulting fromthe interference of the sample arm 422 and the reference arm 423.Multiple images can be typically detected for different positions of thereference arm mirror 425. The exemplary images may be arithmeticallycombined to extract the information from an optical section within thesample.

In another exemplary embodiment of the FFOCM system as depicted in FIG.4B, a broad bandwidth light source 451 can be coupled into a modulatingelement, such as a Michelson interferometer or other interferometer(e.g. Mach-Zehnder, Sagnac) or spectrum altering unit. For the Michelsoninterferometer case, light from the source 451 can be transmitted to abeam splitter 452. Light may then be split into two arms for exemplaryarm A 453 and arm B 455. Light from arm A 453 is transmitted to a mirrorand backreflected back to a beam splitter. Light from arm B 455 islikewise transmitted to a mirror 456, and backreflected back to a beamsplitter 452. The difference between the path length L_(a) in arm A andthe path length L_(b) in arm B, |L_(a)-L_(b)|, can be set to besubstantially equal to the path length difference between the referenceand sample arms in the second interferometer. At least one of the arms Aor B can be configured to change path lengths or produce a phase shiftin the light therein. In one exemplary embodiment, the path length maybe changed by a motion of one of the mirrors or a rapidly scanningoptical delay line. The motion may be actuated by a piezoelectrictransducer, galvanometer, linear motor or the like. Alternatively, pathlength changes may be generated by one of an acousto-optic modulator orelectro optic modulator.

Both reference and sample arm light may be combined at the beamsplitter, and transmitted to another static interferometer with beamsplitter 459, separating light into a reference arm 458 and a sample arm457, respectively. Light from both arms 457, 458 can illuminateobjective lenses 460, 470, respectively, which are substantiallysimilar. In the reference arm 458, the reference objective lens 460 canbe brought to a focus on a reflector 465, which is typically not moving,whereas in the sample arm the sample objective lens 470 focuses thesample arm light on or within the sample 480. The sample 480 or thesample objective lens 470 may be mounted to a stage 481, capable ofmoving the sample 480 in any of three-dimensions, under manual controlor computer control.

The path length difference between the path lengths of the reference arm458 and the sample arm 457 may be substantially equal to |L_(a)-L_(b)|of the first interferometer. Light from reference and sample arms 458,457, respectively, can be combined at a beam splitter 459, and imagedonto a CCD array 490 or array of detectors via a lens 485. A FFOCM imageor data can be generated by a linear combination of images acquired byCCD 490 and while moving or at different positions of mirror 456.Processing, display and storage of FFOCM images is provided by a CPU495. Accumulations or averages are utilized to increase signal to noiseratio.

FIG. 5 depicts an exemplary embodiment of a SECM system configured for afluorescence detection and using a broad bandwidth source. For example,light from the source 500 can be transmitted to a beam splitter 510,which splits light into two paths 515 and 520. Both arms/paths terminateon mirrors 520 and 525, with at least one arm having a path length orphase that changes over time. Light returned from both arms 530 can becoupled to the beam splitter 510 and directed 535 towards a SECM probecontaining a grating or dispersive element 540, an objective lens 550.As discussed herein, the arrangement of the grating and the objectivelens 550 focuses a spectrally encoded line 560 on or within the specimen562 which may be mounted to a three-dimensional stage. Fluorescent lightwithin the sample can be excited by the illuminating light, transmittedback through the objective lens 550, imaged by another lens 565 onto adetector 570. Detected light can be digitized and converted to a line inan image by a processing arrangement (e.g., CPU) 580. Additional linesin the image may be generated at different positions of the beamscanning mechanism 537. Nonlinearities in the moving mirror can becorrected by an exemplary interferometer 521 that has a narrow bandwidthsource that illuminates the same moving mirror 520.\

FIG. 6 depicts a schematic diagram of an exemplary embodiment of an SECMsystem that uses a wavelength tuning source 600. For example, the source600 can be coupled into a circulator/beam splitter 610. According to oneexemplary embodiment, light from the splitter 610 is transmitted via anoptical fiber to a scanner, which alternatively may also contain atelescope lens imaging system that projects the scan axis to the backfocal plane of the objective lens 625. Light from the scanning mechanismis transmitted to a dispersive element 620 (such as a diffractiongrating, prism, GRISM, or DP-GRISM, etc.). Light from 620 is transmittedto an objective lens 625, with preferably a high NA, which can focus thebeam within the sample 635. At any point in time, one wavelength fromthe wavelength swept source 600 can illuminate a distinct portion of thesample. As the wavelength of the swept source 600 changes over time, thebeam can be scanned along a line 630 within the sample 635. Remittedlight from the sample 635 can be transmitted back through the elements625, 620, and 615, respectively, spatially filtered by the optical fiberor a pinhole and transmitted back to the circulator beam splitter 610.Light from the splitter 610 can be directed to a detector 640, anddigitized by a processing arrangement (e.g., CPU) 650, displayed anddigitally stored. A single line in the image is obtained following onefull sweep of the wavelength-tuning source. Lines may be acquired atdifferent positions of the scanning mechanism to form the image.Fluorescent light excited by the wavelength-tuning source 600 remittedfrom the sample can be alternatively detected by a detector 660 to forma fluorescent image.

FIG. 7 depicts a schematic diagram of an exemplary OFDI system. In oneexemplary embodiment of this exemplary OFDI system, a wavelength tuningsource may be coupled to an optical fiber-based circulator 705 and abeam splitter 705. Light from the circulator 705 can be transmitted tothe beam splitter 705, configured to send a majority of light, in thepreferred embodiment, to the sample arm 725. Such split light forwardedto the reference arm 715 can be terminated by a reflector 720, and sentback to the beam splitter 710 and the circulator 705. Light in thesample arm 725 is transmitted to a scanning mechanism 730 and an imaginglens 735 that has a NA sufficiently low to allow cross-sectional imagingof the biological specimen 740. Light is reflected from the referencemirror 720 and the sample 740, recombined at the circulator 705, anddirected by an optical fiber 750 to a detector apparatus 755, which inan exemplary embodiment can contain dual-balanced detectors.

Light is digitized by the detector apparatus 755 and the digital signalis transmitted to a CPU 760. Spectral interference is processed in amanner similar to the processing using the exemplary SD-OCTsystem/procedure, e.g., the background is subtracted, λ-space isconverted to k-space, and an inverse Fourier transform is performed toproduce an A-line. A-lines can be acquired as a function of scanningmechanism position, creating a cross-sectional OFDI image. Alternativeexemplary embodiments known in the art, including the capability toobtain spectral information from the sample by short-time-Fouriertransformation (STFT) of the spectral interference, complex spectraldomain processing, Doppler-sensitive OFDI and polarization-sensitiveOFDI, may be also utilized to extract additional information from thebiological specimen, such as absorption, flow, and birefringence.

FIG. 8 depicts a schematic diagram of an exemplary embodiment of an OCMsystem which utilizes a wavelength tuning/modulated source. For example,a wavelength modulation arrangement 805 may produce a spectral patternon the source, for example, a sinusoidal modulation of the spectrum,which may be altered over time to correspond to spectral interferencemodulation produced by interference between the sample and referencearms. Light from the source 800 and/or the modulation arrangement 805can be coupled into a fiber-optic circulator/beam splitter 810, andsubsequently transmitted to a beam splitter 815 which preferably directsa majority of light to the sample arm 830.

Light in the reference arm 820 is directed towards a reference reflector825 or a transmission element. Light in the sample arm 830 can betransmitted to an x-y scanner, which may comprise one or more ofgalvanometers, resonant scanners, polygon scanners, acousto-opticscanners, electro optic scanners, etc. Light from the scanner can bealternatively transmitted to a telescope 837 and an objective lens 840with preferably a high NA. The objective lens 840 focuses the lightwithin the sample 845, which is alternatively affixed to athree-dimensional stage 847. Light is returned from the sample backthrough the elements 840, 837 and 835 and coupled back into preferablethe core of an optical fiber or pinhole in the sample arm 831 to rejectout-of-focus light. Light is directed to the circulator 810 andtransmitted to a detector 855, digitized and transmitted to a CPU 860.

In one exemplary embodiment, OCM data can be obtained by obtainingA-lines from the sample in a similar manner to the way it is performedusing the exemplary OFDI system and procedure. For example, in theexemplary spectral modulation system and procedure, the detector can besynchronized to the source modulation element 805. Lock-in orsubtraction techniques can be utilized to extract the OCM signal in thiscase. An exemplary image can be generated by acquiring data for eachposition of the x-y scanning mechanism 835. Fluorescent light remittedfrom the sample can be further detected by use of a dichroic mirror orfilter 853 and a second detector 865.

FIG. 9 depicts an exemplary embodiment of an FFOCM system that utilizesa wavelength-tuning/modulated source 900. The light source may be tunedover its bandwidth or alternatively be modulated to contain a spectralmodulation frequency substantially similar the frequency provided byspectral interference modulation of the interferometer. Light from thesource 900 may be coupled into a beam splitter 905, and directed to asample arm 910 and a reference arm 915, respectively, which areterminated by respective objectives 920, 930. The reference armobjective lens 920 focuses reference arm light onto a reflector, whichis subsequently returned to the beam splitter 905. Sample arm light isfocused by 930 onto or within the specimen 935. Light remitted from thesample is combined with the reference arm light at 905, and imaged by alens 940 onto a CCD array 950. Images can be obtained for eachwavelength of the wavelength swept source or different modulationpatterns of the source and arithmetically combined by a CPU 960 toreconstruct an exemplary FFOCM optical section.

According to one exemplary embodiment of the present invention , theexemplary systems described above and alternative exemplary embodimentsthereof may be combined to form a multimodality imaging system. Thisexemplary combination of systems and/or devices can be provided bycreating separate systems, and configuring their optics so that they canobtain images from the same portions of the biological specimen.Different wavelength, scanning, and detection mechanisms may be providedin such combined modality system. Alternatively, the different devicescan be implemented using many common components, which they share toprovide a more efficient, cost-effective apparatus.

FIG. 10 depicts a schematic diagram of a multimodality system accordingto an exemplary embodiment of the present invention that utilizes abroad bandwidth source 1000 and spectrometer 1080 to providesimultaneous and co-registered SD-OCT, OCM, SECM, and fluorescence SECMdata and/or images. For example, light from the broad bandwidth source1000 can be coupled alternatively to a spectral modulation unit 1005.Light from the spectral modulation unit 1005 is coupled into acirculator 1010 and a beam splitter 1015. If a circulator is utilized,light from the circulator 1010 is transmitted to the beam splitter 1015that preferably directs a majority of light to the sample. Light in thereference arm 1020 is transmitted to a reference reflector 1025 that maymove or otherwise change the path length of 1020, and/or which can benon-movable. If the reference arm is allowed to move, conventionaltime-domain OCT (e.g., TD-OCT) arrangement and/or procedures may beimplemented or complex spectral domain may be obtained using theexemplary SD-OCT arrangement and/or procedures using processes that areknown in the art.

Light in the sample arm 1030 is transmitted to a filter/dichroic/WDMapparatus 1035 that transmits the sample arm light in the direction fromthe beam splitter to the sample. Light from 1035 is directed to a beamscanning mechanism 1040 that is capable of scanning the beam in twodirections at high or slow speeds. The beam scanning mechanism 1040 mayalso contain a telescope for imaging the scanners onto the back focalplane of the lens 1055. Light from the scanning mechanism 1040 can betransmitted to a slider 1045 that contains multiple optical elements.For example, when the slider 1045 is positioned at a distinct position,either one or more or a combination of SD-OCT, OCM, SECM and/orfluorescence OCM arrangements/procedures can be implemented. Light fromthe slider 1045 can be transmitted to an objective lens 1055 mounted toa lens turret in one embodiment that is capable of changing objectivelenses. The slider 1045 and/or turret 1050 may be under computer controlfor automatic selection of imaging modality. Light is focused byobjective lens 1055 onto or within the sample 1060, which may be mountedto a computer-controlled three-dimensional translation stage 1065.Reflected light is transmitted back through the apparatus to 1010, whichredirects the light to a spectrometer. Detected reflected light isprocessed to form exemplary SD-OCT, OCM, SECM images using thearrangements and/or procedures described herein.

As shown in FIG. 10, fluorescent light may be redirected to a seconddetector via the filter/dichroic mirror/WDM apparatus 1035 to a seconddetector 1075. Fluorescent light from 1075 is utilized to reconstruct afluorescent confocal image of the biological sample 1060. In the casewhere invisible near-infrared light is utilized, a visible aiming beammay be coupled into the exemplary system, coincident with thenear-infrared light, to allow visualization of the locations of imaging.Alternatively or in addition, a white light image of the specimen underinvestigation may be provided by use of an alternative imaging port onthe microscope. Alternative exemplary embodiments known in the art,including the capability to obtain spectral information from the sampleby short-time-Fourier transformation (STFT) of the spectralinterference, Doppler-sensitive SD-OCT and polarization-sensitiveSD-OCT, may be also utilized to obtain additional information from thebiological specimen, such as, e.g., absorption, flow, and birefringence.

An alternative exemplary multimodality embodiment configured to provideSD-OCT, OCM, SECM, and FFOCM images and data according to the presentinvention at a different wavelength from the other three modalities isdepicted in FIG. 11. In this exemplary embodiment a broad bandwidthsource 1100 is coupled alternatively to a spectral modulation unit 1105.Light from the spectral modulation unit 1105 is coupled into acirculator 1110 and a beam splitter 1115. If the circulator 1110 isutilized, light from the circulator 1110 can be transmitted to the beamsplitter 1115 that preferably directs a majority of light to the sample.Light in the reference arm 1120 is transmitted to a reference reflector1125 that can be stationary and/or may or otherwise change the pathlength of the reference arm 1120. In case the reference arm 1120 isallowed to move, exemplary conventional time-domain OCT (TD-OCT)procedures or complex spectral domain may be utilized for SD-OCT bymethods known in the art. Light in the sample arm 1130 is transmitted abeam scanning mechanism 1135 that is capable of scanning the beam in twodirections at high or slow speeds. The beam scanning mechanism 1135 mayalso include a telescope for imaging the scanners onto the back focalplane of the lens 1160. Light from the scanning mechanism 1135 istransmitted to a dichroic splitter/WDM 1140 that transmits theexcitation light for SD-OCT, OCM, and SECM modalities, and can reflectFFOCM light.

For example, an exemplary FFOCM system similar to that shown in FIG. 3can be coupled into the beam path via 1140. Light from 1140 is directedto a slider 1150 that contains multiple optical elements; when theslider may be positioned at a distinct position, either one or acombination of SD-OCT, OCM, SECM or FFOCM is provided. Light from theslider 1150 is transmitted to an objective lens 1160 mounted to a lensturret 1155 in one embodiment that is capable of changing objectivelenses. The slider 1150 and/or turret 1155 may be under computer controlfor automatic selection of imaging modality. Light is focused by theobjective lens 1160 onto or within the sample 1165, which may be mountedto a computer-controlled three-dimensional translation stage 1170.Reflected light is transmitted back through the apparatus to thecirculator 1110, which redirects the light to a spectrometer. Detectedreflected light may be processed to form exemplary SD-OCT, OCM, SECMimages by methods described herein. FFOCM light may be redirected to theFFOCM system 1175 via the filter/dichroic mirror/WDM apparatus 1140.

In the case where invisible near-infrared light is utilized, a visibleaiming beam may be coupled into the exemplary system shown in FIG. 11,coincident with the near-infrared light, to allow visualization of thelocations of imaging. Alternatively or in addition, a white light imageof the specimen under investigation may be provided by use of analternative imaging port on the microscope. Alternative exemplaryembodiments known in the art, including the capability to obtainspectral information from the sample by short-time-Fouriertransformation (STFT) of the spectral interference, Doppler-sensitiveSD-OCT and polarization-sensitive SD-OCT, may be also utilized toextract additional information from the biological specimen, such as,e.g., absorption, flow, and birefringence.

FIG. 12 depicts an exemplary embodiment of an arrangement of slidersthat may be utilized for the multimodality imaging according to thepresent invention. For example, optical elements can be contained in ahousing 1200 that may be translated manually, or under computer orautomatic control. Each slider position can terminate in differentslider positions 1205, 1210, 1230, 1260 that provide one or more imagingmodalities. The slider position 1205, 1210, 1230, 1260 may be coupled tothe objective lens turret. In one exemplary embodiment, the sliderposition 1205 contains no optical elements (air) or optical elementwindows. In this exemplary configuration, the microscope is configuredto perform FFOCM. For the slider position 1210, a lens apparatus 1212and 1213 can be configured to expand the beam and illuminate a DP-GRISMcontaining two prisms 1215 and 1225 that surround a transmission grating1220. This exemplary configuration provides an ability to perform theSECM imaging. Exemplary OCM procedures can also be conducted in thisposition using a scanning mechanism that scans the spectrally-encodedline across the sample. For the slider position 1230, a lens apparatus1240, 1250 can be configured to image beam angle, with or without beammagnification. This slider position 1230 can provide imaging usingexemplary SDOCT procedures. For the slider position 1260, a lensapparatus 1270, 1280 is configured to expand the scanned beam to allowimaging using the exemplary OCM procedures.

While certain embodiments of the multimodality imaging systems haveutilized a broad bandwidth source, exemplary embodiments of combinedsystems can also include wavelength tuning/modulated sources and singleor multiple detector configurations, and such exemplary embodiment isshown in FIG. 13. For example, in FIG. 13, a wavelength tuning/modulatedsource 1300 is coupled into a circulator 1305 and a beam splitter 1310.If a circulator is utilized, light from the circulator 1305 istransmitted to a beam splitter 1310 that preferably directs a majorityof light to the sample. Light in the reference arm 1315 is transmittedto a reference reflector 1320 that may be stationary, and may orotherwise change the path length of 1315. In case the reference arm isallowed to move, conventional exemplary time-domain OCT (TD-OCT)procedures may be provided or complex spectral domain may be utilizedfor implementing OFDI modalities by methods known in the art. Light inthe sample arm 1325 is transmitted to a filter/dichroic/WDM apparatus1330 that transmits the sample arm light in the direction from the beamsplitter to the sample. Light from 1330 is directed to a x-y beamscanning mechanism 1335 that is capable of scanning the beam in twodirections at high or slow speeds.

The beam scanning mechanism 1335 may also include a telescope forimaging the scanners onto the back focal plane of the lens 1353. Lightfrom the scanning mechanism 1335 is transmitted to a slider 1340 thatcontains multiple optical elements; when the slider is positioned at adistinct position, either one or a combination of OFDI, OCM, SECM orfluorescence OCM modalities can be provided. Light from the slider 1340is transmitted to an objective lens 1353 mounted to a lens turret 1350in one embodiment that is capable of changing objective lenses. Theslider 1340 and/or turret 1350 may be manual, under computer control forautomatic selection of imaging modality. Light is focused by objectivelens 1353 onto or within the sample 1355, which may be mounted to acomputer-controlled three-dimensional translation stage 1360. Reflectedlight is transmitted back through the apparatus to 1305, which redirectsthe light to a detector apparatus 1380 suitable for detecting OFDI,wavelength tuning OCM or SECM signals, images and/or data. Detectedreflected light is processed by a CPU 1385 to form exemplary OFDI, OCM,SECM images by methods described above.

Fluorescent light may be redirected to a second detector via thefilter/dichroic mirror/WDM apparatus 1330 to a second detector 1370.Fluorescent light from 1370 is utilized to reconstruct a fluorescentconfocal image of the biological sample 1355. In the case whereinvisible near-infrared light is utilized, a visible aiming beam may becoupled into the system, coincident with the near-infrared light, toallow visualization of the locations of imaging. Alternatively or inaddition, a white light image of the specimen under investigation may beprovided by use of an alternative imaging port on the microscope.Alternative embodiments known in the art, including the capability toobtain spectral information from the sample by short-time-Fouriertransformation (STFT) of the spectral interference, Doppler-sensitiveSD-OCT and polarization-sensitive SD-OCT, may be also utilized toextract additional information from the biological specimen, such asabsorption, flow, and birefringence.

Another exemplary multimodality embodiment of a system according to thepresent invention which is configured to provide OFDI, OCM, SECM, andFFOCM images, data and other information, where FFOCM signal is providedat a different wavelength from the other three modalities, is depictedin FIG. 14. In this exemplary embodiment, a wavelength tuning source1400 is coupled alternatively to a spectral modulation unit 1405. Lightfrom the modulation unit 1405 is coupled into a circulator 1410 and abeam splitter 1415. If the circulator 1410 is utilized, light from thecirculator 1410 is transmitted to the beam splitter 1415 that preferablydirects a majority of light to the sample. Light in the reference arm1420 is transmitted to a reference reflector 1425 that may bestationary, or can move or otherwise change the path length of 1420. Incase the reference arm 1420 is allowed to move, conventional time-domainOCT (TD-OCT) procedures and modalities may be provided or complexspectral domain may be obtained for the OFDI data by methods known inthe art. Light in the sample arm 1430 is transmitted a beam scanningmechanism 1435 that is capable of scanning the beam in two directions athigh or slow speeds. The beam scanning mechanism 1435 may also contain atelescope for imaging the scanners onto the back focal plane of the lens1465. Light from the scanning mechanism 1435 is transmitted to adichroic splitter/WDM 1445 that transmits the excitation light for OFDI,OCM, and SECM, but reflects FFOCM light.

An exemplary FFOCM system similar to the system(s) of FIG. 3 and/or FIG.4 can be coupled into the beam path via the dichroic splitter/WDM 1445.Light from the dichroic splitter/WDM 1445 is directed to a slider 1455that contains multiple optical elements; when the slider 1455 ispositioned at a distinct position, either one or a combination of OFDI,OCM, SECM or FFOCM data and/or images is provided. Light from the slider1455 is transmitted to an objective lens 1465 mounted to a lens turret1460 in one exemplary embodiment that is capable of changing theobjective lenses. The slider 1455 and/or a turret 1460 may be undercomputer control for an automatic selection of imaging modality. Lightcan be focused by objective lens 1465 onto or within the sample 1470,which may be mounted to a computer-controlled three-dimensionaltranslation stage 1475.

Reflected light is transmitted back through the apparatus to 1410, whichredirects the light to a spectrometer. Detected reflected light isprocessed to form OFDI, OCM, SECM images by methods described herein.FFOCM light may be redirected to the FFOCM system 1450 via thefilter/dichroic mirror/WDM apparatus 1445. In the case where invisiblenear-infrared light is utilized, a visible aiming beam may be coupledinto the exemplary system, coincident with the near-infrared light, toallow visualization of the locations of imaging. Alternatively or inaddition, a white light image of the specimen under investigation may beprovided by use of an alternative imaging port on the microscope.Alternative exemplary embodiments known in the art, including thecapability to obtain spectral information from the sample byshort-time-Fourier transformation (STFT) of the spectral interference,Doppler-sensitive OFDI and polarization-sensitive OFDI may be alsoutilized to extract additional information from the biological specimen,such as absorption, flow, and birefringence.

In another exemplary embodiment of the present invention, the microscopecan be configured to allow imaging from both sides of the sample. Forexample, SDOCT, SECM and OCM procedures can be performed from above thesample, and FFOCM procedures may be performed with the imaging lensilluminates the sample from below. In such exemplary configuration, thesample can be mounted between a microscope slide and a thin cover glass,to allow imaging from both sides.

The exemplary systems described herein can provide a multimodalityimaging of biological specimens in a variety of different formats,speeds, resolutions, fields of view, and contrast mechanisms. Each imagedata set may be two- or three-dimensional, and may be co-registered tothe data sets of the other respective imaging modalities. Computerprocessing methods known in the art may be utilized to display thedifferent data sets in a variety of different imaging formats includingthree-dimensional volume visualization, four-dimensionalrepresentations, and processed two-, three- and four-dimensional datasets, where the processing apparatus is configured to highlightimportant areas of interest. Any one or more datasets may be displayedwith respect to the other and a comprehensive, all-inclusive dataset maybe derived from a combination of the individual data sets. Quantitativeinformation may be derived from the data sets in their two-, three-, andfour-dimensional contexts. Image data may also be combined withconventional fluorescent or brightfield images of the biologicalspecimen.

EXAMPLES

Provided below are examples conducted to investigate using exemplarymultiple imaging modalities according to the present invention to imagethe developing Xenopus laevis heart.

Exemplary methods

Bench-Top Exemplary OCT and OFDI Systems

In the exemplary TDOCT configuration, axial ranging is performed by useof low coherence reflectometry where the individual depth points areprobed sequentially in time. A broad bandwidth (50 nm) source centeredat 1.3 μm was used, providing an axial resolution of ˜10 μm in tissue(n=1.4). The frame rate was 20 per second (2 kHz A-line rate, 100×500pixels).

Exemplary OFDI procedures and systems can use a frequency domainreflectometry in which all depth points are acquired simultaneously.This technique provides a several-hundred-fold improvement insignal-to-noise ratio (SNR) as described in M. A. Choma et al.“Sensitivity advantage of swept source and Fourier domain opticalcoherence tomography,” Optics Express 11, pp 2183-2189 (2003); and S. H.Yun et al., “High-speed optical frequency-domain imaging,” OpticsExpress 11, pp. 2953-2963 (2003). The exemplary OFDI systems andprocedures can use a rapidly swept, wavelength tunable laser as a lightsource. An extended-cavity semiconductor laser employing an intracavityspectral filter, as described in M. A. Choma et al. “Sensitivityadvantage of swept source and Fourier domain optical coherencetomography,” Optics Express 11, pp 2183-2189 (2003), C. Boudoux et al.,“Rapid wavelength-swept spectrally encoded confocal microscopy,” OpticsExpress 13, pp. 8214-8221 (2005).

The laser featured a sweep repetition rate of up to 64 kHz, a widetuning range of 111 nm centered at 1320 nm, and a high average outputpower of 30 mW (7 mW on the tissue). The axial resolution was 10 μm intissue. The system further comprised an acousto-optic frequency shifter(25 MHz) to remove the depth degeneracy inherent in the frequency-domainreflectometry, as described in S. H. Yun et al., “Removing thedepth-degeneracy in optical frequency domain imaging with frequencyshifting,” Optics Express 12, pp. 4822-4828 (2004).Polarization-diversity detection was implemented to eliminatepolarization artifacts in the fiber-based OFDI system. Dual-balancedphotoreceivers were used to improve imaging sensitivity through thereduction of laser intensity noise. The photoreceiver outputs weredigitized with a 2-channel analog-to-digital converter at a samplingrate of 100 MHz with 14-bit resolution.

Exemplary TDOCT and high-speed OFDI configuration were incorporated intoa dissecting light microscope. The scanning system was comprised of acollimating lens (5 mm beam diameter), two synchronized galvanometricscanners for transverse scanning, a focusing lens (50 mm focal length),and a small mirror that deflected the beam downward toward the sample.For exemplary TDOCT and OFDI configuration, the transverse resolutionwas 16 μm with a confocal parameter of 330 μm.

Displacements associated with local cardiac motion were determineddirectly from the volumetric data by subtracting the heart surfacelocations at end diastole from those at end systole on a frame-by-framebasis. Displacement was displayed using a color look up table.Volumetric rendering and three-dimensional visualization wasaccomplished by using OsiriX software.

High-resolution OFDI procedure was performed using a laser source with200 nm tuning range, centered at 1250 nm, in which two semiconductoroptical amplifiers were utilized as the gain media, as described in W.Y. Oh et al., “Wide tuning range wavelength-swept laser with twosemiconductor optical amplifiers,” IEEE Photonics Technology Letters 17,pp. 678-680 (2005). An axial resolution of 4 μm in tissue was achieved.The transverse resolution was 2 μm with NA=0.2 objective lens. Theimaging rate was 40 frames per second with an A-line rate of 20 kHz (500A-lines per frame). Polarization-diversity and dual-balanced detectionwas performed and the photoreceiver outputs were digitized with a2-channel analog-to-digital converter at a sampling rate of 10 MHz with12-bit resolution.

Exemplary FFOCM System

For example, FFOCM is an interferometric technique that utilizestwo-dimensional parallel detection to provide subcellular resolutionimages of reflected light within biological specimens, as described inA. Dubois et al., “Ultrahigh-resolution full-field optical coherencetomography,” Appl Opt 43, pp. 2874-2883 (2004), and A. Dubois et al.,“Three-dimensional cellular-level imaging using full-field opticalcoherence tomography,” Phys Med Biol 49, pp. 1227-1234 (2004). Theexemplary FFOCM system used spatially incoherent broadband light from axenon arc lamp to illuminate the sample and the reference mirror of aLinnik interference microscope using two identical NA=0.3water-immersion microscope objective lenses. Interference images werecaptured with a CMOS area scan camera with spectral response centered at650 nm. The transverse resolutions were 2 μm and axial resolution, 1.1μm. Acquisition time was 2 seconds per frame for a transverse field ofview of approximately 700 μm×700 μm. Three-dimensional data was obtainedby moving the sample through the focus at 1 μm increments. Volumetricrendering and visualization was accomplished by using OsiriX software.

Exemplary SECM System

For example, SECM is a reflectance confocal microscopy technique, whichuses near-infrared light that allows deeper penetration into tissue, asdescribed in R. R. Anderson et al., “The optics of human skin,” J InvestDermatol 77, pp. 13-19 (1981), compared with confocal microscopes thatutilize visible light. Exemplary SECM technique differs fromconventional laser scanning confocal microscopy in that it projectsdifferent wavelengths onto distinct locations on the sample, asdescribed in G. J. Tearney et al., “Spectrally encoded confocalmicroscopy,” Optics Letters 23, pp. 1152-1154 (1998). Rapid acquisitionof spectra returned from the sample enables high-speed reconstruction ofthe image. In the SECM system, as described in C. Boudoux et al., “Rapidwavelength-swept spectrally encoded confocal microscopy,” Optics Express13, pp. 8214-8221 (2005), light from a rapid wavelength tuning source inthe near-infrared (center wavelength=1.32 μm, instantaneous linewidth=0.1 nm, total bandwidth=70 nm, repetition rate up to 15.7 kHz),was collimated onto a diffraction grating (1100 lines per mm) andfocused using a 1.2 NA, 60× objective (Olympus UPlanApo/IR 60×/1.20W). Amultimode fiber was used for signal collection, resulting in 0.9 μmtransverse and 2.5 μm axial resolutions. Images comprised of 500×500pixels were acquired at 10 frames per second. The maximum imaging depthwas limited to the 280 μm working distance of the objective lens.

Specimen Preparation, Ethanol Treatment and Histology

Xenopus laevis frogs were purchased from Nasco (Fort Atkinson,Wisconsin). Animal procedures were performed according to the approvedprotocols of Massachusetts General Hospital Subcommittee on ResearchAnimal Care. Embryos were obtained by in vitro fertilization, incubatedin 0.1× Marc's modified Ringer's medium (MMR)(as described in J. Newportet al., “A major development transition in early Xenopus embryos: 1.Characterization and timing of cellular changes at the midblastulastage,” Cell 30, pp. 675-686, 1982), and staged according to Nieuwkoopand Faber tables. (see P. D. Nieuwkoop and J. Faber, Normal table ofXenopus laevis, Daudin, North-Holland Publishing Company, Amsterdam,1967).

Ethanol treatments were performed in 0.1× MMR (vol/vol), soon after MidBlastula Transition (stage 8.5) (as described in R. Yelin et al.,“Ethanol exposure affects gene expression in the embryonic organizer andreduces retinoic acid levels,” Dev Biol 279, pp. 193-204 (2005).) untilimaging. Prior to in vivo imaging, embryos were anesthetized using 0.02%3-aminobenzoic acid ethyl ester (A-5040, Sigma). For TDOCT and OFDIimaging techniques and systems, embryos were positioned on a 1.5%agarose gel plate with their ventral side facing up, covered by theanesthesia working solution. For imaging with the exemplary SECM system,embryos were placed on a cover slip, lying on their ventral side in ananesthesia buffer, and imaged from below. In vitro imaging by theexemplary FFOCM procedures and/or systems commenced following fixationin MEMFA (0.1M MOPS [pH7.4], 2 mM EGTA, 1 mM MgSO4 and 3.7%formaldehyde) for greater than one hour. Prior to imaging, the fixedembryos were transferred into a Petri dish with 1× PBS (8 gr NaCl, 0.2gr KCl, 1.44 gr Na2HPO4, 0.24 gr KH2PO4), with its ventral side facingup, supported by clay.

Plastic Histology sections (as described in A. M. Glauert, Fixation,Dehydration and Embedding of Biological Specimens., North-HollandPublishing Company Amsterdam, 1986) were obtained after additionalfixation in Karnovsky's Fixative (KII) and embedding in tEpon-812(Tousimis). Sections of 1 μm thick were cut on a Reichert UltracutMicrotome and stained with methylene blue/toluidine blue in boratebuffer (Tousimis). Paraffin sections (5 μm thickness) were stained withHematoxylin & Eosin.

Exemplary Results

Four-Dimensional Imaging of Embryonic Heart with OFDI Techniques in Vivo

Rapid volumetric imaging of the beating heart enables the evaluation ofthree-dimensional morphology and function during the cardiac cycle.Compared with TDOCT, which provides cross-sectional imaging in vivo (asshown in FIGS. 15 a and 15 b), the exemplary OFDI system and procedurecan image at much higher frame rates, making four-dimensional imaging ofthe beating heart without cardiac gating possible. Volumetric OFDIimages of the Xenopus heart (stage 49) were acquired at a rate of 20three-dimensional data sets per second (as shown in FIGS. 15 c-15 g). Atend systole, the use of the OFDI procedure demonstrated that theventricle was at its smallest volume; the volumes of the atrium andtruncus arteriosus (TA) were conversely at their maxima (as shown inFIGS. 15 c and 15 d). At end diastole, the ventricle was dilated to itsgreatest volume, whereas the volumes of the atrium and TA were at theirminima (as shown in FIGS. 15 e and 15 f). A three-dimensional renderingof the heart (as shown in FIG. 15 g), taken from the four-dimensionaldata set, corresponds to a brightfield photograph of the same heartfollowing its dissection (as shown in FIG. 15 h).

High-Resolution OFDI Procedure on Embryonic Heart in Vivo

While the exemplary OFDI system was capable of four-dimensional imaging,there are cases where higher resolution is required to identify subtlemorphological and functional abnormalities. In order to increaseresolution, OFDI cross-sections of a stage 49 Xenopus heart wereobtained in vivo (as shown in FIGS. 15 i-15 m) using a broadband (e.g.,200 nm) wavelength-swept source, as described in W. Y. Oh et al., “Widetuning range wavelength-swept laser with two semiconductor opticalamplifiers,” IEEE Photonics Technology Letters 17, pp. 678-680 (2005)Compared to the 16 μm transverse and 10 μm axial resolutions of thepreviously described TDOCT and OFDI procedures and systems, thetransverse and axial resolutions of high resolution OFDI results were 2μm and 4 μm, respectively. Details within the three-chamber Xenopusheart can be clearly resolved with the high-resolution OFDI proceduresand systems, including atrioventricular valve dynamics (as shown inFIGS. 15 i-15 k), ventricular contractions, and trabecular dynamics(FIG. 15 m). Individual blood cells can also be seen, flowing from theatrium to the ventricle through the atrioventricular valve (as shown inFIG. 15 k).

High-Resolution Three-Dimensional Imaging of Embryonic Heart Using FFOCMProcedures in Vitro

Exemplary FFOCM procedures and systems offer the capability to imagemicrostructure of the embryonic heat with nearly isotropic cellularlevel resolution. Volumetric FFOCM images spanned a field of view of700×700×1000 μm (axial). The transverse and axial resolutions were 2 μmand 1.1 μm, respectively. Acquisition time was 2 seconds for a single enface section, and 33 minutes for the entire volume. Exemplary FFOCMsections of the Xenopus heart (stage 49) allow visualization ofventricular trabeculae (as shown in FIGS. 16 a and 16 c), the spiralvalve (as shown in FIGS. 16 b and 16 d, see arrows), and the partialatrial septum (as shown in FIG. 16 d, see arrow head) with greaterdetail than generated using the exemplary TDOCT or OFDI procedures orsystems. Partially transparent volumetric rendering of the heart (asshown in FIGS. 16 e-16 h), reveals the looping-compression structurewith the angled TA (as shown in FIG. 16 e), the aortic arches (as shownin FIGS. 16 f and 16 g), and the thin wall of the atrium (as shown inFIGS. 16 g and 16 h), in their three-dimensional context. Cut-away viewsof (as shown in FIG. 16 e) show fine three-dimensional internalstructures, including the trabeculae (as shown in FIGS. 16 i and 16 j)and the atrioventricular valve (as shown in FIG. 16 k). A magnified viewof the atrioventricular valve shown (as shown in FIG. 16 l) next to acorresponding histology section of the same embryo (as shown in FIG. 16m), demonstrates its bicuspid morphology.

High-Speed Imaging of Embryonic Heart with SECM Procedures in Vivo

Exemplary SECM procedures and systems provide a transverse resolutioncomparable to those associated with FFOCM, but at higher frame rates,enabling microscopy of the heart in vivo. The Xenopus myocardium (stage49) was imaged in vivo using the exemplary SECM procedures and systemsat a frame rate of 10/s, a field of view of 220×220 μm, and transverseand axial resolutions of 1.2 and 6 μm, respectively. The maximumpenetration depth was 280 μm. Exemplary of the same tadpole (stage 49)visualized by TD-OCT (as shown in FIGS. 15 a and 15 b) and FFOCM (asshown in FIGS. 16 a-16 m) procedures and systems, show the thin cusps ofthe atrioventricular valve (as shown in FIG. 17 a), approximately 280 μmbelow the ventral surface, and parts of the ventricle and TA (as shownin FIGS. 17 c), containing individual blood cells within theintratrabecular spaces. SECM images correlated well with correspondinghistology sections (as shown in FIGS. 17 b and 17 d). A series of framesfrom a different tadpole (stage 47), demonstrates the spiral valve as itcloses (as shown in FIG. 17 e) and opens (as shown in FIGS. 17 f and 17g), regulating blood flow, seen at the single-cell level, from the TA tothe aortic bifurcation. Blood cells are also apparent within thetrabeculae (as shown in as shown in FIGS. 17 h). Intracellular featureswithin individual myocytes that may represent nuclei and organelles canbe observed.

Aneurismal Dilatation in the Xenopus Embryo

In one of the embryos (stage 47), a protrusion emanating from the TAwall has been identified. SECM sections obtained in vivo at twodifferent depths (as shown in FIGS. 18 a and 18b), reveal its saccularshape, its location with respect to spiral valve, as well the flow ofindividual blood cells through the defect. This abnormality was alsoobserved using the exemplary TDOCT procedures and systems in vivo (asshown in FIG. 18 a, see inset). The embryo was then fixed and imagedwith the exemplary FFOCM procedures and systems. An FFOCM section (asshown in FIG. 18 c) and a three-dimensional rendering of the FFOCMvolumetric data set (as shown in FIG. 18 d) show the dilatation in thecontext of the entire heart. Difficult to see under conventionalbrightfield microscopy (as shown in FIG. 18 e), but clearly visualizedusing exemplary TDOCT, FFOCM and SECM procedures and systems, thisprotrusion may represent a saccular aneurismal dilatation of the TA, ina heart that otherwise appeared to have a normal phenotype.

Heart Abnormalities Due to Ethanol Exposure

Cardiovascular malformation can be caused by genetic (as described in K.L. Clark et al., “Transcription factors and congenital heart defects,”Annu Rev Physiol 68, pp. 97-121 (2006)) and teratogenic factors (asdescribed in S. M. Mone et al., “Effects of environmental exposures onthe cardiovascular system: prenatal period through adolescence,”Pediatrics 113, pp. 1058-1069 (2004)). Ethanol is a well-knownteratogen; exposure of human embryo during pregnancy to alcohol(ethanol) is associated with Fetal Alcohol Syndrome (FAS). (See K. L.Jones et al., “Recognition of the fetal alcohol syndrome in earlyinfancy,” Lancet 2, pp. 999-1001 (1973), and J. D. Chaudhuri, “Alcoholand the developing fetus—a review,” Med Sci Monit 6, pp. 1031-1041(2000)). One estimate indicates that 54% of the children with FAS haveheart defects. (See E. L. Abel, Fetal Alcohol Syndrome, MedicalEconomics Books, Oradell, N.J., 1990).

In order to study the teratogenic effect of ethanol on Xenopus heartdevelopment, embryos were exposed to different concentrations of ethanol(0.5%-2.5%) from the mid blastula transition (stage 8.5). (See R. Yelinet al., “Ethanol exposure affects gene expression in the embryonicorganizer and reduces retinoic acid levels,” Dev Biol 279, pp. 193-204(2005)). Siblings developing under the same conditions, but not exposedto ethanol were used as controls. During the developmental process wescreened the heart area of the embryos using the exemplary TDOCTprocedures and systems to identify and qualitatively evaluate the extentof the teratogenic effect. We did not observe morphologic differencesbetween the 0.5% ethanol treated group (n=16) and the control group(n=42). Moderate teratogenic effects, defined as complete maturationwith a substantial change in morphology compared to the controls, wasfound in a minority (25%) of embryos that were exposed to 1% ethanol(n=28), and in a majority (74%) of embryos that were exposed to 1.5%ethanol (n=27). Severe effect, defined as grossly abnormal rotation ofthe heart tube and/or incomplete maturation, was found in all theembryos in the 2.0% and 2.5% groups (n=17, n=7, respectively). Cardiacmotion was evident in all embryos, even those with the most severemalformations.

Using the exemplary TDOCT procedures and systems, a tadpole (stage 48)has been selected from each of the control, 0.5%, 1.5%, and 2.0% ethanoltreated groups to demonstrate typical phenotypes (as shown in FIGS. 19a-19 d). It was determined that the four tadpoles' hearts were inadvanced developmental stages by identifying the existence of a partialatrial septum (as shown in FIGS. 19 a-19d, see right images, septamarked by arrows) and an atrioventricular valve. The TDOCT imagesprovided the first indication of damaged looping in the 1.5% and 2.0%groups. Further observed were lower TDOCT signal from within theventricle in the 1.5% and 2.0% groups, which may be attributed todiminished blood flow in these embryos. Photographs of the tadpoles,taken in vivo from the ventral aspect, are shown in FIGS. 19 e-19 h.

Three-dimensional rendering of data acquired with the exemplary FFOCMsystems and procedures in vitro allowed evaluation of myocardialstructure at high-resolution, revealing the similarity between thecontrol and the 0.5% tadpoles and clearly showing defective heart tubelooping in the tadpoles from the 1.5% and 2.0% groups (as shown in FIGS.19 i-19l). Sections through the FFOCM volumetric data sets demonstratedsmaller, distorted TA's and spiral valves (marked by arrows) in the 1.5%(as shown in FIG. 19 o) and 2.0% embryos (as shown in FIG. 19 p)compared with the control (as shown in FIG. 19 m) and the 0.5% (as shownin FIG. 19 n) embryos. Pericardial edema was present in the 1.5% and2.0% groups (as shown in FIGS. 19 o, 19 p, 19 s and 19 t), compared withcontrol and 0.5% groups. Ethanol also affected the ventricle; thedeveloped trabeculae in the control (as shown in FIG. 19 q) and 0.5% (asshown in FIG. 19 r) hearts contrast the less developed trabeculae in the1.5% group (as shown in FIG. 19 s) and the large ventricular cavity withsparse, stunted trabeculae in embryos exposed to 2.0% ethanol (as shownin FIG. 19 t). Corresponding histological sections confirmed some of ourfindings, including the less developed trabeculae (as shown in FIGS. 19u-19 x) in embryos with the greater ethanol exposure.

Discussion of Exemplary Results

A common paradigm in developmental biology research is to manipulate thegenotype and monitor the phenotype. Morphology is an important aspect ofthe phenotype. In the heart, even slight morphological and dynamicalabnormalities may be critical for proper myocardial function. An abilityto identify subtle morphological and dynamical variations in two andthree dimensions can significantly improve the sensitivity of thisparadigm.

In the Xenopus tadpole, heart structures such as the myocardium wall,septum and valves may only be a few cells thick. Evaluating themorphological phenotype not only requires resolving such finestructures, but also the capability to visualize these microscopicfeatures within the beating heart, where typical displacement velocitiesare on the order of 1 mm/sec. If the imaging speed is sufficiently high,three-dimensional images of the embryo heart can be obtained atdifferent times within the cardiac cycle. This exemplaryfour-dimensional imaging could allow reliable measurements of dynamicphysiological parameters, such as stroke volume and ejection fraction,as well as valve opposition, stiffness and modularity, which have closeanalogs in human pathophysiology. High resolution and high speed are notthe only requirements for effective imaging of the heart. In the Xenopusembryo, the heart extends from between 200 μm and 800 μm beneath theventral surface. An effective imaging method should therefore also becapable of imaging at these depths without substantial loss of signaland resolution.

The morphology of the developing Xenopus laevis heart has been studiedin vitro and described in detail, using three-dimensional rendering ofhistology sections. (See T. J. Mohun et al., “The morphology of heartdevelopment in Xenopus laevis,” Dev Biol 218, 74-88 (2000)). Forhistologic studies, however, sample preparation and sectioning makepreserving structural fidelity difficult. As a result, imaging of intactembryos in their natural environment is preferred. Structural imaging ofthe heart in vivo has been demonstrated using a variety of non-invasiveimaging modalities such as micro-MRI (see D. L. Kraitchman et al., “Invivo magnetic resonance imaging of mesenchymal stem cells in myocardialinfarction,” Circulation 107, pp. 2290-2293 (2003), and F. Wiesmann etal., “Developmental changes of cardiac function and mass assessed withMRI in neonatal, juvenile, and adult mice,” Am J Physiol Heart CircPhysiol 278, pp. H652-657 (2000)), micro-CT (see M. Malyar et al.,“Relationship between arterial diameter and perfused tissue volume inmyocardial microcirculation: a micro-CT-based analysis,” Am J PhysiolHeart Circ Physiol 286, pp. H2386-2392 (2004), and C. T. Badea et al.,“4-D micro-CT of the mouse heart,” Mol Imaging 4, pp. 110-116 (2005)),ultrasound (see S. Srinivasan et al., “Noninvasive, in utero imaging ofmouse embryonic heart development with 40-MHz echocardiography,”Circulation 98, pp. 912-918 (1998)), and PET (see L. W. Dobrucki et al.,“Molecular cardiovascular imaging,” Curr Cardiol Rep 7, pp. 130-135(2005), and L. Stegger et al., “Monitoring left ventricular dilation inmice with PET,” J Nucl Med 46, pp. 1516-1521 (2005)).

Optical techniques enable imaging of the embryonic heart at higherresolution. Confocal microscopy has been used to image early Xenopusheart development, in vitro (as described in S. J. Kolker et al.,“Confocal imaging of early heart development in Xenopus laevis,” DevBiol 218, pp. 64-73 (2000)), and to study the role of intracardiac fluidforces in zebrafish embryonic cardiogenesis, in vivo (as described in J.R. Hove et al., “Intracardiac fluid forces are an essential epigeneticfactor for embryonic cardiogenesis,” Nature 421, pp. 172-177 (2003)).Doppler TDOCT procedures and systems were used to study blood flow inthe Xenopus tadpole, allowing quantitative velocity measurements underthe tissue surface. (See J. R. Hove et al., “Intracardiac fluid forcesare an essential epigenetic factor for embryonic cardiogenesis,” Nature421, pp. 172-177 (2003), and V. X. D. Yang, M. L. Gordon, E. Seng-Yue etal., “High speed, wide velocity dynamic range Doppler optical coherencetomography (Part II): Imaging in vivo cardiac dynamics of Xenopuslaevis,” Optics Express 11, pp. 1650-1658 (2003)). Due to its limitedimaging speed, three-dimensional heart imaging using TDOCT has primarilyonly been previously demonstrated in vitro. (See S. A. Boppart et al.,“Noninvasive assessment of the developing Xenopus cardiovascular systemusing optical coherence tomography,” Proc Natl Acad Sci U S A 94, pp.4256-4261 (1997), T. M. Yelbuz et al., “Optical coherence tomography: anew high-resolution imaging technology to study cardiac development inchick embryos,” Circulation 106, pp. 2771-2774 (2002), and W. Luo etal., “Three-dimensional optical coherence tomography of the embryonicmurine cardiovascular system ” Journal of biomedical optics 11, 021014(2006).

Gating or post-acquisition synchronization techniques have been employedto circumvent the limited speed of conventional imaging methods,enabling the reconstruction of three-dimensional images of embryo heartsat different stages in the cardiac cycle. (See M. W. Jenkins et al., “4Dembryonic cardiography using gated optical coherence tomography,” OpticsExpress 14, pp. 736-748 (2006). M. Liebling et al., “Four-dimensionalcardiac imaging in living embryos via postacquisition synchronization ofnongated slice sequences,” J Biomed Opt 10, 054001 (2005). For some ofthe experiments, we utilized TDOCT as it was more readily available inour laboratory, however the exemplary OFDI procedures and systems werecapable of providing all of the functionality of the exemplary TDOCTprocedures and systems at much higher speeds. The exemplary OFDIprocedures and systems provided real-time, true four-dimensional imagingof a beating heart without requiring cardiac gating and was found to beuseful for assessing myocardial wall displacement during the cardiaccycle (as shown in FIGS. 15 c-15 f).

By modifying the OFDI light source, we were also able to conductreal-time cross-sectional imaging with higher axial resolution (4 μm),enabling visualization of valve dynamics (as shown in FIGS. 15 i-15 k)and single-cell blood flow. For subcellular-level resolution imaging ofthe embryonic heart, we investigated the use of the exemplary FFOCM andSECM procedures and systems. The FFOCM modality was found to be capableof providing high quality three-dimensional imaging with isotropiccellular (1-2 μm) resolution. The SECM modality demonstrated comparableresolution to the FFOCM modality , but was capable of imaging at higherspeeds, enabling visualization of myocyte, blood, and valve motion invivo at the subcellular level. Table 1 summarizes the differentcapabilities of each procedure, highlighting their complementary nature.TABLE 1 Comparison of endogenous-contrast modalities for optical imagingof the embryonic heart. Cells shaded in gray denote the imagingtechnologies with the best transverse resolution, axial resolution, andframe rate characteristics. OCT* FFOCM SECM Transverse resolution 2-16μm   2 μm 0.9 μm Axial resolution 4-10 μm 1.1 μm 2.5 μm Speed [framesper 10-1000 0.5 10 second] Three dimension in Yes No No vivo (4D)Applications Architectural Whole organ Subcellular dynamics microscopicdynamics morphology*Includes TDOCT and OFDI modalities.

The large penetration depth of the exemplary TDOCT and FFOCM proceduresand systems allowed imaging of the heart through pericardial edema thatdeveloped as part of the ethanol teratogenic phenotype. Our preliminaryresults suggest that ethanol interferes with the process of heartlooping (FIGS. 19 i-l), in agreement with a study in quail. (See W. O.Twal et al., “Retinoic acid reverses ethanol-induced cardiovascularabnormalities in quail embryos,” Alcohol Clin Exp Res 21, pp. 1137-1143(1997)). The reduction in TA size that is reported in this work waspredicted by Cavierres and Smith, (see M. F. Cavieres et al., “Geneticand developmental modulation of cardiac deficits in prenatal alcoholexposure,” Alcohol Clin Exp Res 24, pp. 102-109 (2000)), but notobserved. It is believed that the less developed ventricular trabeculaedescribed here (as shown in FIGS. 19 q-t) have not been previouslydeveloped. Since in Xenopus and zebrafish (Danio rerio), the ventriculartrabeculae serve as a functional equivalent of the His-Purkinje system(see D. Sedmera et al., “Functional and morphological evidence for aventricular conduction system in zebrafish and Xenopus hearts,” Am JPhysiol Heart Circ Physiol 284, pp. H1152-1160 (2003)), .a determinationof less developed trabeculae could be associated with the slower heartrate that has been reported in ethanol treated quail (see W. O. Twal etal., “Retinoic acid reverses ethanol-induced cardiovascularabnormalities in quail embryos,” Alcohol Clin Exp Res 21, pp. 1137-1143(1997)), and zebrafish embryos (see J. Bilotta et al., “Ethanol exposurealters zebrafish development: a novel model of fetal alcohol syndrome,”Neurotoxicol Teratol 26, pp. 737-743 (2004)). Interruption of activeblood circulation due to ethanol treatment (W. O. Twal et al., “Retinoicacid reverses ethanol-induced cardiovascular abnormalities in quailembryos,” Alcohol Clin Exp Res 21, pp. 1137-1143 (1997), and X. Wang etal., “Japanese medaka (Oryzias latipes): developmental model for thestudy of alcohol teratology,” Birth Defects Res B Dev Reprod Toxicol 77,pp. 29-39 (2006)) may explain the loss of signal from within the heartcavities, which is also consistent with the determinations.

Despite their relatively high penetration depth, none of theconventional optical imaging procedures could image the heart at theonset of cardiac organogensis (heart tube formation, stage 29), due tohigh scattering at these earlier stages. The initiation of cardiacmovements (stage 35), however, was observed and detailed structuralimages at the onset of chamber formation (around stage 40) were obtainedas the embryo became optically transparent. Especially for the FFOCM andSECM modalities, it was difficult to match histology to the microscopydata sets. The embryos were quite fragile when processed and embedded,making preservation of morphology challenging. Furthermore, imagesshould be registered to histology with a precision on the order of 10μm, which is difficult to achieve with conventional sectioningtechniques.

For the imaging procedures according to exemplary embodiments of thepresent invention, contrast was generated by endogenous scattering.Still, molecular imaging may be important for relating gene and proteinexpression to phenotype. Thus, the exemplary systems and methodsdescribed herein can be used for imaging fluorescent labels andmolecular species. It has been described that fluorescence imaging canbe conducted via spectral encoding by modification of the source anddetection electronics. (See J. T. Motz et al., “Spectral- andfrequency-encoded fluorescence imaging,” Opt Lett 30, pp. 2760-2762(2005)). The same principles used in fluorescence SECM procedures andsystems can likewise be utilized for endoscopic two-photon and secondharmonic imaging. With the coherent detection used in the exemplaryTDOCT, OFDI, and FFOCM procedures and systems, it may be difficult todirectly detect fluorescence. However, several molecular contrastmethods have already been described for the OCT modality. (See C. Yang,“Molecular contrast optical coherence tomography: a review,” PhotochemPhotobiol 81, pp. 215-237 (2005) and S. A. Boppart, et al., “Opticalprobes and techniques for molecular contrast enhancement in coherenceimaging,” J Biomed Opt 10, 41208 (2005)).

The natural contrast optical imaging modalities presented in this workallow evaluation of the embryonic heart from different vantage points.Combining OFDI, SECM, and FFOCM modalities can leverage their strengths(see Table 1), and provide a ability for obtaining a more comprehensivemorphological and functional myocardial phenotype. This multi-modalityparadigm can be extended to other systems and animal models as well.Since these non-invasive imaging techniques do not alter the specimen,they can be used sequentially or in parallel. Furthermore, while we haveused separate imaging systems in this work, there is no fundamentalbarrier preventing their combination into one imaging system that uses asingle wavelength swept source. (See S. H. Yun et al., “High-speedoptical frequency-domain imaging,” Optics Express 11, pp. 2953-2963(2003); C. Boudoux et al., “Rapid wavelength-swept spectrally encodedconfocal microscopy,” Optics Express 13, pp. 8214-8221 (2005); and W. Y.Oh et al., “Wide tuning range wavelength-swept laser with twosemiconductor optical amplifiers,” IEEE Photonics Technology Letters 17,pp. 678-680 (2005)).

The foregoing merely illustrates the principles of the invention.Various modifications and alterations to the described embodiments willbe apparent to those skilled in the art in view of the teachings herein.Indeed, the arrangements, systems and methods according to the exemplaryembodiments of the present invention can be used with any OCT system,OFDI system, SD-OCT system or other imaging systems, and for examplewith those described in International Patent ApplicationPCT/US2004/029148, filed Sep. 8, 2004, U.S. patent application Ser. No.11/266,779, filed Nov. 2, 2005, and U.S. patent application Ser. No.10/501,276, filed Jul. 9, 2004, the disclosures of which areincorporated by reference herein in their entireties. It will thus beappreciated that those skilled in the art will be able to devisenumerous systems, arrangements and methods which, although notexplicitly shown or described herein, embody the principles of theinvention and are thus within the spirit and scope of the presentinvention. In addition, to the extent that the prior art knowledge hasnot been explicitly incorporated by reference herein above, it isexplicitly being incorporated herein in its entirety. All publicationsreferenced herein above are incorporated herein by reference in theirentireties.

1. An apparatus comprising: at least one first arrangement configured toprovide first data associated with a first signal received from at leastone region of at least one sample based on a first modality, and seconddata associated with a second signal received from the at least onesample based on a second modality which is different from the firstmodality, wherein the at least one first arrangement is furtherconfigured to receive third data associated with a reference; and atleast one second arrangement configured to generate further data basedon the first, second and third data.
 2. The apparatus according to claim1, wherein the first modality is spectrally-encoded confocal microscopy.3. The apparatus according to claim 1, wherein the second modality isflorescence imaging.
 4. The apparatus according to claim 1, furthercomprising a microscope arrangement which is associated with the firstand second arrangements.
 5. The apparatus according to claim 1, furthercomprising a beam-scanning arrangement which is configured to forwardelectromagnetic radiation to the at least region.
 6. The apparatusaccording to claim 1, wherein the at least one second arrangementgenerates at least one of (i) a two-dimensional image or (ii) athree-dimensional image as a function of the further data.
 7. Theapparatus according to claim 1, wherein the first and second data areobtained substantially simultaneously.
 8. The apparatus according toclaim 1, wherein the first and second data are associated withapproximately the same location on the at least one sample.
 9. Theapparatus according to claim 1, wherein at least one of the first andsecond data is obtained using another one of the first and second data.10. The apparatus according to claim 1, wherein the first and secondarrangements are provided in at least one of a probe or a singleenclosure.
 11. The apparatus according to claim 1, wherein the first andsecond arrangements include common components.
 12. The apparatusaccording to claim 11, wherein the common components are provided in atleast one of a source arrangement or a detector arrangement.
 13. Theapparatus according to claim 1, wherein the at least one firstarrangement is configured to obtain spectral encoding microscopyinformation.
 14. The apparatus according to claim 1, wherein the atleast one first arrangement is configured to obtain at least one ofbright field, dark field, phase contrast, polarization, epireflectanceor reflectance microscopy information.
 15. The apparatus according toclaim 1, further comprising a further arrangement which is configured tochange from the first modality to the second modality.
 16. The apparatusaccording to claim 1, wherein the at least one first arrangement isconfigured to obtain optical coherence tomography information associatedwith a signal provided by a source arrangement having a plurality ofwavelengths, and further comprising a plurality of detectors configuredto detect a spectral interference between the second and third signalsas a function of the wavelengths.
 17. The apparatus according to claim1, wherein the at least one first arrangement is configured to obtainoptical coherence tomography information associated with a signalprovided by a source arrangement whose wavelength varies over time. 18.The apparatus according to claim 1, wherein the at least one firstarrangement is further configured to receive third data associated witha reference, and the at least one second arrangement is configured togenerate the further data further the third data
 19. The apparatus ofclaim 1, further comprising: at least one third arrangement configuredto control at least one of the first arrangement based on at least oneof the first data or the second data.
 20. The apparatus of claim 1,further comprising: at least one fourth arrangement configured togenerate an image based on the first and second data.
 21. The apparatusof claim 1, further comprising: at least one fifth arrangementconfigured to generate at least one first image based on the first dataand at least one second image based on the second data, wherein thefirst and second images are associated with one another as a function ofthe first and second data.
 22. The apparatus according to claim 1,wherein the at least one first arrangement is configured to obtainoptical coherence tomography information.
 23. The apparatus according toclaim 1, wherein the at least one first arrangement is configured toobtain optical frequency domain interferometry information.
 24. Anapparatus comprising: at least one first arrangement configured toprovide first data associated with a first signal received from at leastone region of at least one sample based on a first modality, second dataassociated with a second signal received from the at least one samplebased on a second modality which is different from the first modality,and least one third data associated with a second signal received fromthe at least one sample, wherein each of the at least one third data isbased on a further modality which is different from the first modalityand the second modality; and at least one second arrangement configuredto generate further data based on the first, second and third data. 25.The apparatus according to claim 24, wherein the at least one firstarrangement is configured to obtain optical coherence tomographyinformation.
 26. The apparatus according to claim 24, wherein the atleast one first arrangement is configured to obtain optical coherencemicroscopy information.
 27. The apparatus according to claim 24, whereinthe at least one first arrangement is configured to obtain full fieldoptical coherence microscopy information.
 28. An apparatus comprising:at least one first arrangement configured to provide first dataassociated with a first signal received from at least one region of atleast one sample based on a first spectral-encoding modality, and seconddata associated with a second signal received from the at least onesample based on a second non-spectral-encoding modality; and at leastone second arrangement configured to generate further data based on thefirst and second data.
 29. A method comprising: providing first dataassociated with a first signal received from at least one region of atleast one sample based on a first modality, and second data associatedwith a second signal received from the at least one sample based on asecond modality which is different from the first modality; receivingthird data associated with a reference; and generating further databased on the first, second and third data.
 30. A method comprising:providing first data associated with a first signal received from atleast one region of at least one sample based on a first modality,second data associated with a second signal received from the at leastone sample based on a second modality which is different from the firstmodality, and least one third data associated with a second signalreceived from the at least one sample, wherein each of the at least onethird data is based on a further modality which is different from thefirst modality and the second modality; and generating further databased on the first, second and third data.
 31. A method comprising:providing first data associated with a first signal received from atleast one region of at least one sample based on a firstspectral-encoding modality, and second data associated with a secondsignal received from the at least one sample based on a secondnon-spectral-encoding modality; and generating further data based on thefirst and second data.